Protocol

 

Authors

A. Anandan, A. Mahender and S. K. Pradhan

Author Affliations

Central Rice Research Institute, Cuttack, Odisha, India

Overview

The protocol outlined here is to measure whole plant area of seedling to assess the early vigour using digital image and Image J software.

Background

Plant growth occurs by the process of cell division and elongation. Growth depends on availability of assimilates, plant hormones and other substances along with a conducive environment. Growth can be measured for whole plant as total dry matter or plant height and organ level. Estimating growth helps to assess the production potential of a crop and this has already been measured by utilizing the mathematical principles and widely used to understand the yield potential of crop plants. Here an approach has been explained to measure the whole plant area at seedling stage to assess early seedling vigour (ESV) using ImageJ.

Materials/Equipment

Digital camera and computer
Image J software (open-source: http://rsbweb.nih.gov/ij/download.html)
Potted plant
Measuring ruler (used as reference when analyzing image)
Dark colour cloth (used as back drop while imaging)
Small raised platform or table
High-intensity artificial light (halogen lamp)

Units, terms, definitions

mm2 = square millimeters

Procedure

Step1:

– Spread dark colour cloth over the convenient raised platform or table and use the same cloth as backdrop. Therefore, during photograph background should have uniform colour. Place high-intensity artificial light towards the table to get uniform light around the table. Block any strong wind flow or fan, wind is not advisable for this process. Make sure that plant/leaves are not moving.

Step 2:

– Place the potted plant over the table and make sure there is some scale indicator in your image. Better, place a ruler just above the pot, i.e. bottom of the plant and should be visible in the photograph.

– You may include label with details about your plant.

– Now take the image of a plant from three different angles, top, right and left side of the plant. Maintain uniform distance between plant and camera, pixel numbers and light for whole of the experiment. Then, download images and save to computer.

Step 3:

– Open Image J software and select open the desired photograph.

– Select rectangular or oval selection tool to crop the image to have desired portion alone. Now with that tool, mark the desired area and again open image menu and select “crop” to have selected portion.

– Open the image menu again and select “adjust” then select “color threshold”. Adjust the sliding bars of hue, saturation and brightness until your leaf and stem portions are highlighted in red.

– Now set scale to calculate the desired area of your image. Select straight line tool from the tool bar, draw a line across the ruler of known distance, e.g. 10 mm. Open the analyse menu and select “set scale”, type 10 into the “known distance” box, “mm” into the unit of length and select the global box (selecting global, indicates the same measuring scale for consecutive images).

– Open the analyse menu and select “set measurement”, then confirm the selection of area, standard deviation and decimal place as “3”.

– To analyse the whole plant area, select all leaves and stem portion in the image with wand (tracing) tool by holding shift key.

– Open the analyse menu again and select “analyse particles”, a dialogue box opens. In drop down menu of “show”, select “outlines” and click ok.

– A new window opens with the selected portion of plants and other area highlighted by red colour with outline alone.

Step 4:

– Now by using wand (tracing) tool, select the multiple spots one by one by holding shift key and go for measuring them.

– Open the analyse menu and select “measure”. New window appears with results corresponding to the selected area (whole plant area (mm2)) and standard deviation.

– Similarly, measure the area of top view image, right and left side image of the plant. Now, the area of the whole seedling can be obtained by getting mean value of all three images of a genotype by using excel spreadsheet.

– By adopting this protocol, absolute values of plant growth rate based on whole plant area (WPA) between two intervals can be studied to find ESV genotype in any field crops.
AGR (WPA) = (A2-A1/t2-t1) mm2 day-1
Where, AGR is absolute growth rate, A1 and A2 are the whole plant area at times t1 and t2 respectively.